The development of type 2 diabetes is accompanied by a progressive decline in β-cell mass and function. Vildagliptin, a dipeptidyl peptidase 4 inhibitor, is representative of a new class of antidiabetic agents that act through increasing the expression of glucagon-like peptide-1. The protective effect of this agent on β cells was studied in diabetic mice. Diabetic pancreatic β cell-specific C/EBPB transgenic (TG) mice exhibit decreased β-cell mass associated with increased apoptosis, decreased proliferation, and aggravated endoplasmic reticulum (ER) stress. Vildagliptin was orally administered to the TG mice for a period of 24 weeks, and the protective effects of this agent on β cells were examined, along with the potential molecular mechanism of protection. Vildagliptin ameliorated hyperglycemia in TG mice by increasing the serum concentration of insulin and decreasing the serum concentration of glucagon. This agent also markedly increased β-cell mass, improved aggravated ER stress, and restored attenuated insulin/IGF1 signaling. A decrease in pancreatic and duodenal homeobox 1 expression was also observed in β cells isolated from our mouse model, but this was also restored by vildagliptin treatment. The expression of C/EBPB protein, but not mRNA, was unexpectedly downregulated in vildagliptin-treated TG mice and in exenatide-treated MIN6 cells. Activation of the GLP1 pathway induced proteasome-dependent C/EBPB degradation in β cells as the proteasome inhibitor MG132 restored the downregulation of C/EBPB protein by exenatide. Vildagliptin elicits protective effects on pancreatic β cells, possibly through C/EBPB degradation, and has potential for preventing the progression of type 2 diabetes.
Pancreatic β cell failure is a fundamental pathogenic feature of type 2 diabetes, and the progressive decline in β cell function and mass over the course of type 2 diabetes development is well known (Weyer et al. 1999, Butler et al. 2003, Rhodes 2005, Wajchenberg 2007). This occurs regardless of intervention with insulin and/or antidiabetic agents (UK Prospective Diabetes Study (UKPDS) Group 1998a,b). Recent studies have revealed that both attenuated β cell function, and the loss of β-cell mass contribute to the pathogenesis of type 2 diabetes, which has drawn attention to the need for agents capable of protecting β cells.
Dipeptidyl peptidase 4 (DPP4) inhibitors form a new class of antihyperglycemic agents that can be used for the treatment of type 2 diabetes. These agents act by increasing the incretin hormone glucagon-like peptide-1 (GLP1), which stimulates insulin secretion (Holst & Deacon 2004). In addition to this action, GLP1 and its analogs have a variety of other biological effects, including the suppression of glucagon secretion and food intake, as well as the inhibition of gastric emptying (Drucker & Nauck 2006). Furthermore, GLP1 and its analogs have been shown to enhance β-cell mass by increasing the proliferation and decreasing the apoptosis of β cells in animal models (Xu et al. 1999, Wang & Brubaker 2002, Li et al. 2003, Stoffers 2004). By contrast, despite clear evidence of the effects of GLP1 and its analogs, only a small number of reports to date have examined whether DPP4 inhibitors also enhance β-cell mass (Mu et al. 2009, Cho et al. 2011, Duttaroy et al. 2011) or whether the increase in GLP1 levels elicited by DPP4 inhibition leads to the suppression of serum glucagon concentrations (Balas et al. 2007, Ahren et al. 2010).
Endoplasmic reticulum (ER) stress is thought to play an important role in the development of β cell failure in type 2 diabetes (Harding & Ron 2002). We have recently shown that the accumulation of transcription factor C/EBPB in pancreatic β cells contributes to β cell failure by enhancing susceptibility to ER stress (Matsuda et al. 2010). In our previous study, we established two lines of transgenic (TG) mice specifically overexpressing C/EBPB in pancreatic β cells, in which C/EBPB was expressed approximately ten- and three-fold more relative to C57BL/6J mice. These mice exhibited decreased β-cell mass as a result of increased apoptosis and reduced cellular proliferation, and aggravated ER stress was also observed in the β cells of these mice (Matsuda et al. 2010). As a result, both lines of TG mice exhibited hyperglycemia along with decreased plasma insulin concentration in a manner dependent on the level of C/EBPB expression (Matsuda et al. 2010). The first line with a tenfold increase in C/EBPB expression exhibited blood glucose levels of >400 mg/dl in the fed state, while the second line with a threefold increase showed ∼200 mg/dl. In this study, the second line of TG mice was administered the DPP4 inhibitor vildagliptin for up to 24 weeks to examine whether this agent could exert a protective effect on β cells by preserving β-cell mass. The molecular mechanism by which vildagliptin exerted beneficial effects in TG mice was also examined.
Materials and methods
Pancreatic β cell-specific C/EBPB TG mice of the C57BL/6J background were generated as described previously by inserting the C/EBPB gene under control of the rat insulin promoter (Matsuda et al. 2010). Animals were maintained in a 12 h light:12 h darkness cycle and fed normal chow from weaning (3-week old), also as previously described (Kido et al. 2000, Hashimoto et al. 2005). All experiments used male mice, and WT and C/EBPB-TG mice were grouped and housed with either regular water or water continuously supplemented with 0.6 mg/ml vildagliptin (a gift from Novartis Institutes for Biomedical Research, Cambridge, MA, USA) from 4 weeks to 24 weeks of age. Blood glucose and plasma insulin concentrations were determined as described elsewhere (Kido et al. 2000, Hashimoto et al. 2005), while plasma glucagon levels were measured using a RIA kit (Millipore, Inc., Billerica, MA, USA). Plasma GLP1 levels were measured using a GLP1 ELISA kit (Shibayagi, Gunma, Japan). This study was performed according to the guidelines of the Animal Ethics Committee of Kobe University Graduate School of Medicine.
Oral glucose tolerance tests
Mice were deprived of food for 16 h before the oral administration of glucose (1.5 mg/g body weight). Blood was collected immediately before and at 15, 30, 45, 60, and 120 min following administration.
Real-time RT-PCR analysis
Total cellular RNA was extracted from islets isolated from control and C/EBPB-TG mice using an RNeasy kit (Qiagen). Real-time RT-PCR analysis of the total RNA pooled from four to five animals of each genotype was performed as described previously (Matsuda et al. 2010). Values were normalized to cyclophilin mRNA levels and expressed as fold induction. The primers (sense and antisense) used for gene amplification were as follows: 5′-ACCGGGTTTCGGGACTTGA-3′ and 5′-GTTGCGTAGTCCCGTGTCCA-3′ for mouse C/EBPB and 5′-CAGACGCCACTGTCGCTTT-3′ and 5′-TGTCTTTGGAACTTTGTCTGCAA-3′ for cyclophilin.
Infection with a retrovirus encoding C/EBPB
Platinum-E (PLAT-E) ecotropic packaging cells were transfected with the retroviral vector pMX-C/EBPB (encoding mouse C/EBPB) with the use of Lipofectamine 2000 Reagent (Invitrogen). Recombinant retroviruses released into the culture medium were harvested 48 h after transfection and added to MIN6 cells cultured in DMEM supplemented with 15% FBS. After culturing for 8 h, the infected cells were subjected to selection in medium containing puromycin.
Western blot analysis
Lysates of isolated islets and C/EBPB-overexpressed MIN6 cells were prepared as described previously (Kido et al. 2000, Hashimoto et al. 2005) and probed with antibodies for C/EBPB, C/EBP homologous protein (CHOP) (Santa Cruz Biotechnology, Inc.,), phosphorylated c-jun, phosphorylated eIF2α, phosphorylated c-jun N-terminal kinases, the phospho-Thr308 form of Akt, the phospho-Ser473 form of Akt, the cleaved form of caspase-3, phosphorylated protein kinase-like ER kinase (PERK), phosphorylated cAMP response element binding (CREB), proliferating cell nuclear antigen (PCNA; Cell Signaling, Danvers, MA, USA), insulin receptor substrate 2 (IRS2) (Upstate, Lake Placid, NY, USA), β-actin (Sigma–Aldrich), and pancreatic and duodenal homeobox 1 (Pdx1; provided by T Nakamura, Shiga University of Medical Science). Quantitative analysis of the blots was performed using Multi Gauge Version 3.0 software (Fujifilm, Tokyo, Japan) and normalized to β-actin levels.
Incubation with exenatide and MG132 in MIN6 cells
C/EBPB-overexpressed MIN6 cells were incubated with 100 nM exenatide for the indicated time. C/EBPB-overexpressed MIN6 cells were pretreated with 20 μM MG132 for 30 min before exenatide incubation.
Immunostaining and morphometric analysis
Following removal, the pancreas was immersed in Bouin's solution, embedded in paraffin, and sliced into 4 μm thick sections, which were stained with antibody to insulin, glucagon, and PCNA (Dako, Carpinteria, CA, USA). Immune complexes were detected with secondary antibodies conjugated with either Cy3 or FITC (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Quantification of β-cell mass was performed as described previously (Kido et al. 2000, Hashimoto et al. 2005).
Assessment of food intake
The food intake of four mice in each group caged separately was measured for 5 days before and at 8 weeks after vildagliptin treatment.
Islet isolation and insulin content measurement
Islets were isolated from the pancreas by collagenase digestion and handpicked under stereoscopy. For measurement of islet insulin content, ten size-matched islets from each group were solubilized in acid–ethanol solution (75% ethanol, 1.4% HCl) overnight at 4 °C, and insulin concentrations were measured. Data were normalized to total protein content.
All results are expressed as mean±s.e.m. Statistical significance was assessed by ANOVA and, when appropriate, the Student's t-test. A P value of <0.05 was considered statistically significant.
Effect of vildagliptin on blood glucose levels and serum insulin concentrations in pancreatic β cell-specific C/EBPB TG mice
We first examined plasma GLP1 level after 12 weeks of vildagliptin treatment. GLP1 levels in untreated TG mice showed a decreased tendency compared with control mice (Fig. 1A). Vildagliptin increased the GLP1 level by 2.4-fold in control mice and by fourfold in TG mice, although the increase in control mice did not reach statistical significance (Fig. 1A). Vildagliptin treatment did not alter food intake or body weight in either pancreatic β cell-specific C/EBPB TG mice or controls (Fig. 1B and C). As previously reported, blood glucose levels were found to be increased and serum insulin concentrations were decreased in TG mice compared with controls (Fig. 1D and E; Matsuda et al. 2010). Treatment with vildagliptin significantly improved blood glucose levels in TG mice as early as 2 weeks following the initiation of administration. Although the glucose levels of the TG mice did not reach those observed in the controls, this effect was maintained throughout the experimental period for up to 24 weeks (Fig. 1D). Vildagliptin treatment in TG mice was also found to result in a significantly increased serum insulin concentration compared with untreated mice (Fig. 1E). Neither blood glucose level nor serum insulin concentration in controls was affected by vildagliptin treatment (Fig. 1D and E).
Similar results were observed when an oral glucose tolerance test (OGTT) was performed on the TG mice after 8 weeks of vildagliptin treatment. While the TG mice showed increased blood glucose levels associated with decreased insulin levels during the OGTT, vildagliptin treatment resulted in markedly decreased blood glucose levels and increased serum insulin concentrations (Fig. 2A and B). This treatment did not alter either parameter in controls (Fig. 2A and B). The OGTT was also performed after 24 weeks of treatment to examine whether the effects of vildagliptin were maintained for a longer period. After this longer period, improved glucose tolerance was again found to be associated with an increase in serum insulin concentrations in vildagliptin-treated TG mice, suggesting that the effect of vildagliptin on glucose metabolism in these mice is preserved for at least 24 weeks (Fig. 2C and D).
Effect of vildagliptin on serum glucagon concentration in TG mice
GLP1 and its analogs are well known to suppress glucagon secretion of α cells (Drucker & Nauck 2006), although few reports have examined whether the increase in GLP1 level, elicited by DPP4 inhibition, has such an effect (Balas et al. 2007, Ahren et al. 2010). We therefore examined whether vildagliptin treatment had an impact on serum glucagon concentration in TG mice. OGTTs revealed that the basal serum glucagon concentration was significantly increased in vildagliptin-untreated TG mice and was suppressed to a lesser extent following the administration of glucose compared with controls. This suggests that α-cell dysfunction contributes to glucose intolerance in TG mice (Fig. 2E). The basal glucagon concentration and abnormal trend observed after glucose load were significantly improved by vildagliptin treatment in TG mice (Fig. 2E). These effects were also confirmed by the area under the curve during the OGTT (Fig. 2F).
Effect of vildagliptin on β-cell mass in TG mice
We next studied whether vildagliptin treatment resulted in increased β-cell mass in TG mice. Consistent with our previous report (Matsuda et al. 2010), TG mice exhibited a 60% decrease in β-cell mass compared with controls (Fig. 3A and B). A marked increase in β-cell mass was observed in the TG mice after 8 weeks of treatment with vildagliptin (Fig. 3A). When the β-cell mass was quantified, the vildagliptin-treated TG mice showed an 80% increase compared with untreated TG mice (Fig. 3B). We also examined whether the protective effect of vildagliptin on β-cell mass was preserved for a longer period than the initial 8 weeks, and it was found to be maintained in TG mice over the 24 weeks of treatment (Fig. 3C and D). The extent of protection was equivalent between 8 and 24 weeks of treatment (Fig. 3A, B, C, and D). While the insulin content of the islet cells was also found to be significantly decreased in TG mice compared with controls (Fig. 3E), vildagliptin treatment significantly increased the insulin content by ∼2.5-fold in the TG mice (Fig. 3E). We also examined the α cell mass because serum glucagon concentration was increased in TG mice and because vildagliptin treatment improved this alteration in these mice. α cell mass in TG mice showed an increased tendency compared with control mice, although it did not reach statistical significance (Fig. 3A and C, and data not shown). Furthermore, vildagliptin treatment affected α cell mass in neither TG mice nor control mice (Fig. 3A and C, and data not shown).
To further examine the mechanism involved in the increased β-cell mass observed in the TG mice following vildagliptin treatment, β cell proliferation and apoptosis were evaluated. The number of PCNA-positive cells in untreated TG mice was found to be decreased relative to controls (Fig. 4A and B), while vildagliptin partially, but significantly, increased the number of PCNA-positive cells in TG mice (Fig. 4A and B). Although the abundance of PCNA was also found to be decreased in the islets of TG mice, levels were again restored following vildagliptin treatment (Fig. 4C). When apoptosis was assessed, upregulation of CHOP protein and increased abundance of the cleaved form of caspase-3 were observed in the islet cells of TG mice, although these apoptosis markers were significantly inhibited by treatment with vildagliptin (Fig. 5A and C). These results suggest that both increased β cell proliferation and decreased β cell apoptosis contribute to the increased β-cell mass observed following vildagliptin treatment in TG mice.
Effect of vildagliptin on gene expression and signaling pathways in the islets of TG mice
To clarify the molecular mechanism by which vildagliptin ameliorates β cell failure in TG mice, alterations in the expression or phosphorylation status of various genes were examined in islets isolated from the TG mice. First, we checked ER stress signaling, which was composed of the PERK, IRE1a, and ATF6a pathways. Consistent with our previous report (Matsuda et al. 2010), the phosphorylation levels of PERK and eukaryotic translation initiation factor 2A (eIF2α) were increased in untreated TG mice (Fig. 5A and C). IRE1a signaling was also increased in untreated TG mice as shown by the increase in the spliced form of XBP1, which is a downstream target of IRE1a (Fig. 5E and F). Vildagliptin treatment suppressed activation of these pathways in TG mice (Fig. 5A and C). GRP78 expression, which is regulated mainly by the ATF6a pathway, was not altered in TG mice or by vildagliptin treatment (data not shown). Similarly, the increased expression of proteins related to apoptosis, such as CHOP and cleaved caspase-3, observed in TG mice was substantially normalized following vildagliptin treatment, in a manner consistent with the increase in β-cell mass in TG mice (Fig. 5A). In addition, while TG mice exhibited a reduction in the phosphorylation of CREB, a transcription factor known to be activated by GLP1 in a cAMP-dependent manner, vildagliptin treatment reversed this decreased phosphorylation (Fig. 5B and D). Given that CREB regulates IRS2 expression (Jhala et al. 2003), which is a key component of insulin signaling in β cells, and that insulin/IGF1 signaling in β cells plays a pivotal role in maintaining β-cell mass (Withers et al. 1998, Kulkarni et al. 1999, Kubota et al. 2000, Hashimoto et al. 2006, Ueki et al. 2006), we also examined the alteration of insulin/IGF1 signaling in the islets of TG mice. As expected, these mice showed reduced IRS2 expression and attenuated Akt phosphorylation, while vildagliptin treatment increased both IRS2 expression and Akt phosphorylation (Fig. 5B and D). Finally, the PDX1 transcription factor is also essential for regulating β cell development and function (McKinnon & Docherty 2001). While PDX1 expression was markedly reduced in TG mice, vildagliptin almost completely restored PDX1 expression (Fig. 5B and D). Given that insulin/IGF1 signaling also regulates PDX1 expression in β cells (Kitamura et al. 2002), the upregulation of PDX1 expression by vildagliptin is thought to be attributable to the activation of insulin/IGF1 signaling via the cAMP/CREB pathway. C/EBPB expression in TG mice was unexpectedly decreased by vildagliptin treatment, although the mRNA expression of C/EBPB was not altered. This indicates that vildagliptin can elicit posttranscriptional modifications of C/EBPB.
Effect of the GLP1 analog exenatide on gene expression and signaling pathways in C/EBPB overexpressing β cells
We next sought to examine whether the protective effect of vildagliptin on β cell failure in TG mice occurs as a direct result of the activity of this drug on β cells. MIN6 cells were infected with a retrovirus to induce overexpression of C/EBPB before being treated with the GLP1 analog exenatide. The expression levels and activation of various proteins in these cells were then examined. The phosphorylation of proteins involved in ER stress, such as protein kinase RNA-like ER kinase, eIF2a, and c-jun, as well as the expression of apoptosis-related proteins, including CHOP and cleaved caspase-3, was found to be markedly upregulated by C/EBPB overexpression, although these alterations were reversed by incubation with 10 nM exenatide (Fig. 6A and B). These findings suggest that vildagliptin primarily exerts its protective effect on β cells in TG mice, independent of improved glucose metabolism.
Activation of GLP1 pathway induces proteasome-dependent C/EBPB degradation
Exenatide treatment significantly attenuated C/EBPB expression in MIN6 cells (Fig. 6A and B), which was consistent with the results observed in islets isolated from vildagliptin-treated TG mice (Fig. 7A, B, and C). To study the mechanism by which activation of the GLP1 pathway attenuates the expression of C/EBPB protein, we tested the effect of a proteasome inhibitor, MG132, on exenatide-induced downregulation of C/EBPB. Exenatide again attenuated C/EBPB expression in these cells; however, MG132 restored expression of this protein, suggesting that activation of the GLP1 pathway induces proteasome-dependent degradation of C/EBPB protein in β cells (Fig. 7D).
We used pancreatic β cell-specific C/EBPB TG mice to examine whether vildagliptin exerts a protective effect on β cells. As TG mice manifest aggravated ER stress and decreased β-cell mass associated with increased apoptosis and reduced proliferation that are common pathogenic features observed in type 2 diabetes in humans (Harding & Ron 2002, Butler et al. 2003), we reasoned that TG mice would be a useful model for evaluating the beneficial effects of this agent. Although evidence has shown that incretin hormones such as GLP1 and its analogs can increase β-cell mass in animal models via blockading apoptosis or enhancing proliferation and neogenesis (Xu et al. 1999, Wang & Brubaker 2002, Li et al. 2003, Stoffers 2004), reports of whether DPP4 inhibitors increase β-cell mass have only recently started to emerge. We demonstrated that vildagliptin markedly improves insulin response to glucose and increases β-cell mass, leading to an alleviation of glucose metabolism in TG mice. We also showed that these effects are preserved over a relatively long term. Such observations indicate that vildagliptin could protect against the progressive decline in β cell function and mass over the course of type 2 diabetes in humans.
DPP4 inhibitors increase the circulating level of GLP1 and GIP through inhibiting degradation of these incretin hormones. As activation of the GLP1 pathway by exenatide improves aggravated ER stress and decreases insulin/IGF1 signaling by C/EBP overexpression in cultured β cells, the beneficial effect of vildagliptin observed in TG mice is thought to be attributed mainly to activation of the GLP1 pathway. However, the GIP pathway might also contribute to such an effect because GIP has been reported to protect pancreatic β cells (Widenmaier et al. 2010). The underlying molecular mechanism by which DPP4 inhibitors lead to an increase in β-cell mass is not fully understood. Insulin/IGF1 signaling in β cells has been shown to play a pivotal role in maintaining β-cell mass (Withers et al. 1998, Kulkarni et al. 1999, Kubota et al. 2000, Hashimoto et al. 2006, Ueki et al. 2006). In our study, the reduced β-cell mass observed in the TG mice was associated with attenuated insulin/IGF1 signaling, which could be restored by vildagliptin treatment and the resulting increase in the expression of IRS2. In addition to enhancing calcium-dependent insulin secretion (Holst & Gromada 2004), an increase in intracellular cAMP levels leads to the activation of the CREB transcription factor, which is responsible for the regulation of IRS2 expression in β cells (Jhala et al. 2003). In fact, activation of the GLP1 pathway by vildagliptin enhances CREB phosphorylation in β cells in TG mice. Therefore, the CREB-dependent upregulation of IRS2 expression is likely to be involved in the activation of insulin/IGF1 signaling by vildagliptin. This mechanism is supported by a recent study that demonstrated that IRS2 signaling is required for exenatide to have protective effects in β cells (Park et al. 2006). As PDX1 also plays an essential role in β cell development and the maintenance of mature β cell function (McKinnon & Docherty 2001), upregulation of PDX1 expression by vildagliptin also likely contributes to increased β-cell mass and improved insulin response to glucose elicited by this drug. PDX1 expression is known to be regulated by insulin/IGF1 signaling (Kitamura et al. 2002), and it is thus possible that vildagliptin directly upregulates PDX1 in β cells through activation of the insulin/IGF1 pathway.
ER stress is suggested to play an important role in the development of β cell failure in type 2 diabetes (Harding & Ron 2002). TG mice established in one of our previous studies were shown to manifest pancreatic β cell failure due to aggravated ER stress, such as increased eIF2a and c-jun phosphorylation (Matsuda et al. 2010). A previous study has additionally shown that exenatide directly modulates the ER stress response through induction of ATF4 expression and accelerates recovery from ER stress-mediated translational repression in cultured β cells in a cAMP-dependent manner (Yusta et al. 2006). Furthermore, exendin-4 and forskolin have been shown to protect β cells against free fatty acid- and salubrinal-induced ER stress and apoptosis by enhancing the expression of genes such as BiP (Hspa5), Bcl2, and JunB involved in cellular defenses (Cunha et al. 2009). However, the mechanism by which ER stress is regulated by GLP1 signaling remains unclear. In this study, we showed that activation of GLP1 signaling by either vildagliptin or exenatide improves aggravated ER stress in TG mice as well as in C/EBPB overexpressing β cells. As C/EBPB itself plays important roles in ER stress in β cells, we checked C/EBPB expression in vildagliptin-treated TG mice and in exenatide-treated C/EBPB overexpressing β cells. Unexpectedly, we observed that the expression of C/EBPB protein, but not mRNA, was downregulated in these experiments. We found that activation of the GLP1 pathway induced the proteasome-dependent C/EBPB degradation in β cells as MG132 restored the downregulation of C/EBPB protein by exenatide. Although we could not specify the molecular link between the GLP1 pathway and C/EBPB degradation in this study, GLP1 activation might alleviate ER stress in β cells possibly through inducing C/EBPB degradation. Future studies will be needed to uncover the pathway of GLP1-dependent C/EBPB degradation.
In conclusion, vildagliptin was shown to ameliorate hyperglycemia in TG mice by enhancing β-cell mass. It is likely that both the alleviation of ER stress and the enhancement of insulin signaling in β cells contribute to the beneficial effects of this agent. Collectively, our findings support the potential utility of vildagliptin in the protection of pancreatic β cells in patients with type 2 diabetes.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
This work was supported by a grant for a Grant-in-Aid for Scientific Research from MEXT to Y K (22590981) and a Grant-in-Aid for Young Scientists from MEXT to T M (10015786), a Grant-in-Aid for Young Scientists from MEXT to N H (22790863), and a Novartis research grant to Y K.
Author contribution statement
S Shimizu, M K-K, S-i A, T M, T H, S Seino, and Y K conceived and designed the experiments. S Shimizu, A K, M F, H E, H T, K T, H I, and Y M performed the experiments. S Shimizu, A B, M K-K, S-i A, T M, T H, N H, S Seino, and Y K analyzed the data. S Shimizu, A K, A B, M F, and H E contributed reagents/materials/analysis tools. T H, S Shimizu, and Y K wrote the manuscript.
The authors thank D Kawamori (Osaka University) for excellent technical advice and M Nagano for technical assistance.
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